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BackgroundThe Japanese honeybee,Apis cerana japonica, shows a specific defensive behavior, known as a “hot defensive bee ball,” used against the giant hornet,Vespa mandarinia. Hundreds of honeybee workers surround a hornet and make a “bee ball” during this behavior. They maintain the ball for around 30 min, and its core temperature can reach 46. Although various studies have been conducted on the characteristics of this behavior, its molecular mechanism has yet to be elucidated. Here, we performed a comprehensive transcriptomic analysis to detect candidate genes related to balling behavior.ResultsThe expression levels of differentially expressed genes (DEGs) in the brain, flight muscle, and fat body were evaluated during ball formation and incubation at 46 °C. The DEGs detected during ball formation, but not in response to heat, were considered important for ball formation. The expression of genes related to rhodopsin signaling were increased in all tissues during ball formation. DEGs detected in one or two tissues during ball formation were also identified.ConclusionsGiven that rhodopsin is involved in temperature sensing inDrosophila, the rhodopsin-related DEGs inA. cerana japonicamay be involved in temperature sensing specifically during ball formation.

Honey bees and bumble bees belong to the same family (Apidae) and their workers exhibit a division of labor, but the style of division of labor differs between species. The molecular and neural bases of the species-specific social behaviors of Apidae workers have not been analyzed. Here, we focused on two immediate early genes, hormone receptor 38 (HR38) and early growth response gene-1 (Egr1), and late-upregulated ecdysone receptor (EcR), all of which are upregulated by foraging flight and expressed preferentially in the small-type Kenyon cells of the mushroom bodies (MBs) in the honey bee brain. Gene expression analyses in Bombus ignitus revealed that HR38 and Egr1, but not EcR, exhibited an immediate early response during awakening from CO2 anesthesia. Both premature mRNA for HR38 and mature mRNA for Egr1 were induced during foraging flight, and mRNAs for HR38 and Egr1 were sparsely detected inside the whole MB calyces. In contrast, EcR expression was higher in forager brains than in nurse bees and was expressed preferentially in the small-type Kenyon cells inside the MBs. Our findings suggest that Kenyon cells are active during foraging flight and that the function of late-upregulated EcR in the brain is conserved among these Apidae species.

application/pdf, Odors in female mice induce sexual arousal in male mice. Repeated exposure to female odors attenuates male attraction, which recovers when the odors are removed. The neuronal mechanisms for the recovery of male attraction have not been clarified. In this study, we examined how olfactory systems are involved in the recovery of male attraction to female odors following habituation in mice. Presentation with volatile female odors for 5 min induced habituation in males. To evaluate male attraction to familiar volatile female odors, we measured the duration for investigating volatile female odors from the same female mouse, which was presented twice for 5 min with 1-, 3-, or 5-min interval. Intranasal irrigation with ZnSO4 solution almost completely suppressed investigating behavior, indicating that the main olfactory system is indispensable for inducing the attraction to volatile female odors. In contrast, removal of the vomeronasal organ, bilateral lesions of the accessory olfactory bulb (AOB), or pharmacological blockage of neurotransmission in the AOB did not affect the investigation time at the first odor presentation. However, each one of the treatments decreased the investigation time in the second presentation, compared to that in the first presentation, at longer intervals than control treatment, indicating that the disturbance of neurotransmission in the accessory olfactory system delayed the recovery of the attraction attenuated by the first presentation. These results suggest that the accessory olfactory system facilitates the recovery of the attraction to familiar volatile female odors in male mice. © The Author 2017. Published by Oxford University Press. All rights reserved.

Asian honeybees (Apis cerana) have evolved a thermal collective defense against the sympatric giant hornet (Vespa mandarinia) in which workers surround the predator en masse and produce heat up to 46 °C to kill it. This characteristic behavior is called “hot defensive bee ball formation.” Many studies have described the uniqueness and efficiency of this behavior; however, little attention has been paid to the potential cost to the honeybee. In this study, we focused on potential effects to bee ball-participating honeybees. We compared life expectancy of same-age ball-participating honeybees and nonparticipating honeybees and demonstrated that the life expectancy of the bee ball-participating honeybees was dramatically shortened. The 46 °C exposure also shortened the life expectancy of honeybees with induced expression of the heat shock protein gene, strongly implicating the increased temperature inside the bee ball in the deleterious effect on participating honeybees. We additionally found that bee ball-participating and then short-lived worker honeybees had a tendency to join a subsequent bee ball more aggressively. This tendency could mitigate accumulation of the short-lived worker honeybees within the colony, which otherwise would cause a severe reduction of honeybee colony activity. Social insects have evolved unique anti-predator altruistic behaviors for colony defense. Evaluation of the potential cost of these behaviors provides valuable insight into their evolution. Asian honeybees (Apis cerana) exhibit a sophisticated collective defense (bee ball formation) against the sympatric giant hornet (Vespa mandarinia) whose rigid exoskeleton resists the common stinging attack of the honeybees, utilizing heat. We found that the high temperature inside the ball itself dramatically reduced honeybee longevity. Furthermore, bee ball-experienced honeybees were found to be more likely to engage in subsequent bee ball formation. Our results pointed out unavoidable cost for the honeybee colony associated with the use of heat and a “division of risk” strategy in the bee colony for minimizing the cost.

The present study, aiming to elucidate ion channel mechanisms underlying muscarinic receptor–induced depolarization, has characterized membrane currents induced by carbachol in single guinea-pig urinary bladder myocytes. Application of carbachol to cells that were voltage-clamped at − 50 mV produced an atropine-sensitive, biphasic inward current consisting of an initial peak followed by a smaller sustained phase. Replacing the extracellular Na+ and intracellular Cl− with impermeable tris+ and glutamate−, respectively, demonstrated that the biphasic current is entirely composed of cation currents. Its initial peak phase was abolished by buffering intracellular Ca2+ to a constant level of 100 nM or depleting intracellular Ca2+ stores, and it was mimicked by the Ca2+ releaser caffeine. Ca2+ entry evoked by voltage steps in the sustained phase induced no noticeable change, indicating that this phase of cation current is insensitive to a rise of [Ca2+]i. These results demonstrate that muscarinic receptor stimulation invokes the openings of two types of cation channel, a Ca2+-activated and a receptor-operated type; the former channels are gated by a rise in [Ca2+]i upon intracellular Ca2+ release, and the latter are gated through other muscarinic receptor–coupled signal transduction mechanisms. Keywords:: cation channel, muscarinic receptor, carbachol, bladder smooth muscle, guinea pig

We examined the fail-safe responses against low-dose botulinum intoxication (botulinum neurotoxin serotype A; 0.05 ng/35 g body weight) in electrically activated in vitro phrenic nerve-diaphragm preparations, since sustained ventilation is critical for the prognosis of clinical botulinum intoxication. At 0, 1, 2 and 4 wks after the peritoneal injection of the toxin, both contractility and neurotransmitter release were measured. There was an increase in directly induced twitch force without affecting directly induced tetanus throughout the observation period. Indirectly induced twitch force decreased by 60% at 1 wk, which gradually recovered only during the 4-wk observation period. Spontaneous neurotransmitter release, evaluated as the frequency of miniature end plate potentials, was largely abolished 1 wk after the injection and recovered only slightly during the 4-wk period. The effects on spontaneous release were independent of medium Ca2+ concentration. Evoked release, evaluated as quantal content, was also mostly inhibited at 1 wk, but it recovered to approximately 50% of controls at 4 wks. The recovery of quantal content was more prominent at low medium Ca2+ concentration. These results indicated two functional fail-safe responses that compensate for the acute inhibitory effect of low dose of botulinum toxin on neuromuscular transmission; increased contractility of muscle, and improved efficiency of evoked quantum release. The increased contractility probably reflects remodeling of muscle fiber composition of the diaphragm. The improved efficiency of evoked quantum release probably involves remodeling of voltage-gated Ca2+ channels, intracellular Ca2+ store sites, or transmitter-releasing apparatuses.

Using mutant mice genetically lacking certain subtypes of muscarinic receptor, we have studied muscarinic signal pathways mediating cationic channel activation in intestinal smooth muscle cells. In cells from M2 subtype-knockout (M2-KO) or M3-KO mice, carbachol (100 μm) evoked a muscarinic cationic current (mICat) as small as ∼10% of mICat in wild-type (WT) cells. No appreciable current was evoked in M2/M3 double-KO cells. All mutant type cells preserved normal G-protein–cationic channel coupling. The M3-KO and WT mICat each showed a U-shaped current–voltage (I–V) relationship, whereas the M2-KO mICat displayed a linear I–V relationship. Channel analysis in outside-out patches recognized 70-pS and 120-pS channels as the major muscarinic cationic channels. Active patches of M2-KO cells exhibited both 70-pS and 120-pS channel activity usually together, either of which consisted of brief openings (the respective mean open times Oτ= 0.55 and 0.23 ms). In contrast, active M3-KO patches showed only 70-pS channel activity, which had three open states (Oτ= 0.55, 3.1 and 17.4 ms). In WT patches, besides the M2-KO and M3-KO types, another type of channel activity was also observed that consisted of 70-pS channel openings with four open states (Oτ= 0.62, 2.7, 16.9 and 121.1 ms), and patch current of this channel activity showed a U-shaped I–V curve similar to the WT mICat. The present results demonstrate that intestinal myocytes are endowed with three distinct muscarinic pathways mediating cationic channel activation and that the M2/M3 pathway targeting 70-pS channels, serves as the major contributor to mICat generation. The delineation of this pathway is consistent with the formation of a functional unit by the M2-Go protein and the M3-PLC systems predicted to control cationic channels.

Bovine lactoferrin (bLf) is an iron-binding secretory protein present in breast milk, mucosal secretions, and the secondary granules of neutrophils. Although bLf has multiple functions, including antimicrobial and immunomodulatory activities, its effect on neuronal cells is not fully understood. We report that bLf prevents cell adhesion of PC12 cells and allows them to be cultivated in suspension. PC12 cells normally adhere well to plastic culture plates and show anchorage-dependent cell growth, but we found that soon after adding bLf, they detach from culture plates and begin to grow in suspension. When bLf was removed from the medium, the cells began to re-adhere to the plates. Thus, bLf inhibits cell adhesion and stimulates anchorage-independent growth in PC12 cells. On the other hand, bLf-induced cell suspension growth was not observed when cells were grown on a laminin matrix, suggesting that bLf does not affect integrin-mediated cell adhesion on a laminin matrix. Treatment of cells with heparin or chondroitin sulfate A or C inhibited bLf-induced growth in cell suspension. Furthermore, pretreatment of cells with heparinase and/or chondroitinase prevented direct binding of bLf to the cell membrane. These results suggest that bLf binds to the membrane of PC12 cells via membrane-associated proteoglycans and leads to anchorage-independent growth. Keywords:: bovine lactoferrin, cell adhesion, PC12 cell, proteoglycan, cell suspension

application/pdf, Post-weaning mice fed exclusively milk display low-frequency exploratory behavior [Ishii, T., Itou, T., and Nishimura, M. (2005) Life Sci. 78, 174-179] compared to mice fed a food pellet diet. This low-frequency exploratory behavior switched to high-frequency exploration after a switch from exclusively milk formula to a food pellet diet. Acquisition of the high-frequency exploratory behavior was irreversible. Recently, we demonstrated that the mesencephalic trigeminal nucleus (Me5) is involved in the control of feeding and exploratory behavior in mice without modulating the emotional state [Ishii, T., Furuoka, H., Itou, T., Kitamura, N., and Nishimura, M. (2005) Brain Res. 1048, 80-86]. We therefore investigated whether the Me5 is involved in acquisition of high-frequency exploratory behavior induced by the switch in diet from an exclusively milk formula to food pellets. Mouse feeding and exploratory behaviors were analyzed using a food search compulsion apparatus, which was designed to distinguish between the two behaviors under standard living conditions. Immunohistochemical analysis of immediate early genes indicated that the Me5, which receives signals from oral proprioceptors, is transiently activated after the diet change. The change from low-frequency to high-frequency exploratory behavior was prevented in milk-fed mice by bilateral lesion of the Me5. These results suggest that the Me5 is activated by signals associated with mastication-induced proprioception and contributes to the acquisition of active exploratory behavior.

In order to realize fine processing with a high precision optical system, we have developed a laser processing system with galvanometer scanners and optical lenses using a shrink fitter. The shrink fitter prevents the misalignment of the lenses at the elevated temperature. Therefore fine laser spots can be obtained over the quite wider scanning width, even at elevated temperatures. We examined the possibility of application to laser processing of this lens system using the shrink fitter.Diameters of the lenses used in this study were 160 mm so that the laser spots could be scanned over the width of 100 mm, in both directions of the X-axis and Y-axis. There was large distortion of the laser beam which was inherent to each lenses and can not be removed by mechanical adjustment So we introduced the formula to correct the beam path using the least square method. Finally, we developed the fine laser processing system using shrink fitter.

application/pdf, An exclusively milk formula diet stunted the growth of mice immediately following weaning. Milk-fed mice displayed a low-frequency profile of exploratory behavior, while pellet-fed mice showed high-frequency exploration. In contrast to exploratory behavior, feeding behavior did not differ significantly between milk- and pellet-fed mice. Despite showing low-frequency exploratory behavior, mice on an exclusively milk formula diet showed no difference in behavioral activities analyzed by an automatic hole-board apparatus compared to pellet-fed mice. These results suggest that the growth stunt caused by an exclusively milk formula diet retards the acquisition of active exploratory behavior without affecting the emotional state of mice.

Brain and serum were collected from 120 and 12 free-ranging sika deer (Cervus nippon yesoensis), respectively, from six regions in eastern Hokkaido during controlled hunts in the autumn of 2003. Brains were tested for Neospora caninum and Toxoplasma gondii DNA by polymerase chain reaction (PCR) assays. Antibodies to Toxoplasma gondii were measured by means of a latex agglutination test. No brain tested positive for either type of DNA, and no antibody to Toxoplasma gondii was detected in serum, suggesting a low prevalence of infection with these organisms in free-ranging sika deer from eastern Hokkaido. Further examination of multiple tissues by PCR and serologic surveys will be necessary to confirm this.

application/pdf, The mesencephalic trigeminal nucleus (Me5), which receives input from oral proprioceptors and projects to higher brain regions, is involved in mastication-induced modulation of satiation. To investigate how the Me5 is involved in the control of feeding and exploratory behavior, we examined the effect of bilateral electrolytic lesions of the Me5 on feeding and exploratory behavior in mice. Mouse feeding and exploratory behaviors were analyzed using a food- search-compulsion-apparatus (FSCA), which was designed to distinguish between the two behaviors under standard living conditions. To assess anxiety in mice in an unfamiliar environment, exploratory activity was analyzed in an automated hole-board apparatus. Mice with bilateral Me5 lesions had unique feeding and exploratory behavior profiles in the FSCA compared with sham-operated mice. Me5-lesioned mice spent more time in the food chamber during each trial in the FSCA, but the number of entries into the food chamber was decreased by 40% compared to sham-operated mice. Moreover, Me5 lesions markedly inhibited exploratory behavior, manifested as low-frequency exploration. In spite of the low frequency exploration in the FSCA, Me5 lesions had no effect on various exploratory activities analyzed in the hole-board apparatus, i.e., total locomotor activity, frequency and duration of rearing and head-dipping, and latency to the first head-dipping. These results suggest that the Me5 is involved in the control of feeding and exploratory behavior through its ascending neuronal pathways in mice without modulating the emotional state.

To estimate the genetic background of Japanese wild boars inhibiting the 4 prefectures of Shikoku Island (Kagawa, Tokushima, Kouchi and Ehime), we examined haplotypes of mitochondrial DNA (mtDNA) and genotypes of the nuclear glucosephosphate isomerase-processed pseudogene (GPIP) in 189 wild boars. Of the 8 different mtDNA haplotypes (J5, J10, J12, J13, J15, J19, J20 and E33) detected in Shikoku Island, the first to be identified were J19 and J20 (of Japanese wild boar lineage) and E33 (of European domestic pig lineage). The presence of haplotype E33 indicates local crossbreeding between wild boar and escaped domestic pigs and/or crossbred Inobuta. Four kinds of the GPIP genotypes were detected from 189 wild baors in Shikoku Island, but no European GPIP alleles were found. In 113 wild boars, no antibodies against Classical Swine Fever or Aujeszky's disease were detected, suggesting that they had not been exposed to those pathogens.

Although the antioxidant properties of green, oolong, and black teas have been well studied, antioxidant activity has not been examined in roasted tea. Therefore, in the current studies, we investigated the antioxidant activity of roasted tea in comparison with those of green, oolong, and black teas. Using water extracts of the various teas, we examined the total phenolic content as well as the antioxidant activities, including the reducing power, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, and the inhibition of hemolysis caused by 2,2'-azo-bis(2-amidinopropane) dihydrochloride (AAPH)-induced lipid oxidation in erythrocyte membranes. The roasted tea contained lower levels of total phenolics than green, oolong, or black tea (green tea > oolong tea > black tea > roasted tea). The relative reducing power and DPPH scavenging activity decreased in the following order: green tea > roasted tea > oolong tea > black tea. Also, green tea was more effective against AAPH-induced erythrocyte hemolysis than other teas (green tea>roasted tea = oolong tea = black tea). These results suggest that roasted tea is beneficial to health, in humans, because of its high antioxidant activity.

There are several benefits to a high-fat diet for animals exposed to cold, including improved tolerance to severe cold conditions and increased survival rates in cold environments. It is therefore of interest to examine whether animals exposed to cold will selectively consume lipids. We examined the intake of safflower oil (SO) by rats exposed to cold (4 +/- 2 degrees C) under a feeding condition in which the rats were given free access to SO. Rats exposed to cold consumed more SO than those housed at 25 +/- 2 degrees C. This finding suggests that rats prefer SO in a cold environment. There was no significant difference in the ratio of calories of SO ingested to that of matter (standard laboratory chow plus SO) ingested between rats exposed to cold and those at 25 +/- 2 degrees C. The high SO intake also affected cold tolerance and metabolite kinetics in the rats. Factors that affected the SO intake of rats exposed to cold are also discussed.

In these studies, the authors investigated the effect of propylene glycol (PG) on the cytosolic free Ca2+ concentration ([Ca2+]i) in rat cerebrocortical synaptosomes using the fluorescent Ca2+ indicator fura-2. PG (0.5-5% v/v) increased [Ca2+]i in a concentration-dependent manner. The PG-induced increase in [Ca2+]i was inhibited approximately 50% by the omission of extracellular Ca2+ or the addition of Ni2+ (100 microM). Decrease of extracellular Na+ (6.2 mM) or addition of tetrodotoxin (1 microM), verapamil (10 microM), nifedipine (10 microM), omega-agatoxin IVA (200 nM), omega-conotoxin GVIA (1 microM), or omega-conotoxin MVIIC (1 microM) had no effect on the increase in [Ca2+]i. Also, addition of TMB-8 (100 microM), ryanodine (50 microM) or thapsigargin (1 microM) did not modify the increase in [Ca2+]i in the absence of extracellular Ca2+. These results suggest that PG increases [Ca2+]i in rat cerebrocortical synaptosomes by both stimulating Ca2+ entry through a Ni2+-sensitive pathway and releasing Ca2+ from TMB-8-, ryanodine- and thapsigargin-insensitive Ca2+ stores.

We prospected the antidotes from natural oriental drugs for botulinum neurotoxin type A. The crude extracts of Galla Rhois and Geranii Herba showed a potent antidote activity for botulinum neurotoxin type A. The antidote titer of the crude extracts of black tea was higher than those of Galla Rhois and Geranii Herba. No antidote activity was detected in the crude extracts of the followings: Gambir, Rosae Multiflorae Fructus, Scutellariae Radix, Coptidis Rhizoma, Sophorae Flos, Puerariae Radix, Houttuyniae Herba, Swertiae Herba, Galla Halepensis. Although both Galla Rhois and Galla Halepensis are gall-apples and contain tannin as the main component, Galla Rhois showed the potent antidote activity while Galla Halepensis did not.

Adenosine enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. We found that adenosine increases NGF-induced phosphorylation of extracellular signal-regulated kinase (ERK), but decreases the duration of phosphorylation of p38 mitogen-activated protein (MAP) kinase. Therefore, we further examined the involvement of protein phosphatase in these effects of adenosine. FK506, a specific calcineurin inhibitor, inhibited the enhancing effect of adenosine on the NGF-induced neurite outgrowth and increased the duration of p38 MAP kinase phosphorylation without affecting ERK phosphorylation. These results suggest that adenosine decreases the duration of p38 MAP kinase via calcineurin activation, which contributes to the enhancement of NGF-induced neurite outgrowth. Keywords:: PC12 cell, nerve growth factor, p38 mitogen-activated protein kinase, neurite outgrowth, calcineurin

In Ca2+-free medium, propylene glycol (PG) increases the cytosolic-free Ca2+ concentration ([Ca2+]i) in rat cerebrocortical synaptosomes. In the current studies, the authors investigated the intracellular source of this Ca2+. PG (0.5-5% v/v) dose-dependently increased [Ca2+]i in Ca2+-free medium. The increase in [Ca2+]i was completely inhibited by pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP, 10 microM). The inhibitory effect of CCCP was dependent on the concentration and the pretreatment time. These results suggest that, in Ca2+-free medium, PG increases [Ca2+]i in rat cerebrocortical synaptosomes by releasing Ca2+ from mitochondrial stores.

The aim of the present study was to elucidate the mechanism of the protective effect of black tea extract, the thearubigin fraction, against the neuromuscular blocking action of botulinum neurotoxin types A, B, and E. The effects of thearubigin fraction extracted from a black tea infusion were examined on the neuromuscular blocking action of botulinum neurotoxin types A, B, and E in mouse phrenic nerve-diaphragm preparations and on the binding of these toxins to rat cerebrocortical synaptosomes. Botulinum neurotoxin type A (1.5 nM), B (6 nM), or E (5 nM) abolished indirect twitches in mouse phrenic nerve-diaphragm preparations within 50, 90, 90 min., respectively. Thearubigin fraction mixed with each toxin blocked the inhibitory effect of the toxins. The specific binding of [125I]botulinum neurotoxin type A, B, or E to rat cerebrocortical synaptosomes was inhibited by mixing iodinated toxin with thearubigin fraction. The elution profile of [125I]botulinum neurotoxin type A, B, or E on Sephadex G-50 column chromatography was different from that of toxin mixed with thearubigin fraction. These findings indicate that thearubigin fraction protects against the neuromuscular blocking action of botulinum neurotoxin types A, B, and E by binding with the toxins.

紅茶から抽出したテアルビジン分画の,生ハム抽出液に添加したボツリヌス神経毒素に対する効果を確認した.テアルビジン分画は,ボツリヌス神経毒素が神経終末細胞表面へ結合する前に混合することにより濃度依存的にに毒素の作用を阻止した.B型毒素の経口毒性を調べるため,毒素と生ハム由来の上清液との混合試料をマウスに経口投与した.本毒素は濃度依存的にマウスに運動麻痺作用を発現した.さらにテアルビジン分画の抗毒素能を調べるたあ,B型毒素,テアルビジン分画と生ハム上清液との混合試料をマウスに経口投与した.テアルビジン分画はマウスの運動麻痺を濃度依存的に阻止した.テアルビジン分画の食品への応用性を調べるため,テアルビジン分画に浸漬した生ハム試料を用いて官能試験を実施した.生ハムは熱湯抽出したテアルビジン分画に浸漬すると食味の悪化を呈した.しかしテアルビジン分画からタンニンなどの夾雑物を除去することにより,その抗毒素能は高まるとともに食味の悪化を防げると予想した.

application/pdf, Mouse N1E-115 cells grown on a laminin matrix exhibit neurite outgrowth in response to serum deprivation. Treatment of cells with an antibody against beta (1) integrin inhibits neurite outgrowth. Thus, beta (1) integrin is involved in the neuritogenesis of N1E-115 cells on a laminin matrix. Integrin-linked kinase (ILK), a recently identified cytoplasmic serine/threonine protein kinase that binds to the cytoplasmic domain of beta (1) integrin, has an important role in transmembrane signal transduction via integrins. We report that ILK is expressed in N1E-115 cells, the expression levels of which are constant under both normal and differentiating conditions. A stable transfection of a kinase-deficient mutant of ILK (DN-ILK) results in inhibition of neurite outgrowth in serum-starved N1E-115 cells grown on laminin. On the other hand, a transient expression of wild type ILK stimulated neurite outgrowth. The ILK activity in the parental cells was transiently activated after seeding on the laminin matrix, whereas that in the DN-ILK-transfected cells was not. These results suggest that transient activation of ILK is required for neurite outgrowth in serum-starved N1E-115 cells on laminin. Under the same conditions, p38 mitogen-activated protein (MAP) kinase, but neither MAP kinase/extracellular signal-regulated kinase kinase (MEK) nor extracellular signal-regulated kinases (ERK), was transiently activated after N1E-115 cell attachment to laminin, but not in the DN-ILK-expressed cells. The time course of p38 MAP kinase activation was very similar to that of ILK activation. Furthermore, a p38 MAP kinase inhibitor, SB203580, significantly blocked neurite outgrowth. Thus' activation of p38 MAP kinase is involved in ILK-mediated signal transduction leading to integrin-dependent neurite outgrowth in N1E-115 cells.

Angiotensin II (Ang II) and atrial natriuretic peptide (ANP) may be involved in local regulation of the oviductal contraction during the estrous cycle. Thus, the in vitro effects of Ang II and ANP on the secretion and contraction of bovine oviduct during the follicular, postovulatory, and luteal phases were investigated. An in vitro microdialysis system (MDS) was utilized to determine the intraluminal release of prostaglandins (PGs), Ang II, and endothelin-1 (ET-1) from the bovine oviducts as well as to observe the effect of Ang II and ANP on the local secretion of these substances. The basal release of PGs, ET-1, and Ang II was higher (P , 0.05) during the follicular and postovulatory phases than during the luteal phase. Stimulation by infusion of Ang II (10 26 M) or ANP (10 27 M) into the MDS was carried out for 4 h between 4 and 8 h of incubation. In the oviducts from the follicular and postovulatory phases, the infusion of ANP increased the release of Ang II, but not of ET-1. Infusion of Ang II stimulated the release of ET-1. Both Ang II and ANP increased PGE 2 and PGF2a release. In the contraction study, direct administration of Ang II (10 27 M) or ANP (10 28 M) into the medium during the follicular and postovulatory phases increased the amplitude of oviductal contraction. In contrast, these substances did not show any effect in the contraction and secretion of oviducts from cows during the midluteal phase. These results indicate that during the periovulatory period, Ang II and ANP stimulate the contractile amplitude of the oviduct in vitro. In addition to their direct action on oviductal contraction, Ang II may activate oviductal secretion of ET-1 and PGs. Likewise, ANP stimulates oviductal secretion of PGs and Ang II. Hence, the overall results suggest the existence of a functional endothelinangiotensin-ANP system in the bovine oviduct during the periovulatory period, which may regulate the oviductal contraction to ensure maximum efficiency of gamete/embryo transport through the oviduct. hormone action, oviduct, ovum pick-up/transport

The present study investigated the effects of hibernation and hibernating body temperature (10°C) on the relative changes that may occur in adrenergic and purinergic perivascular neurotransmission of the golden hamster. The hindlimb resistance vessels and the tibial artery of age-matched controls, cold exposed controls and hibernated hamsters were examined by pharmacological and electrophysiological techniques. At 34°C, electrical field stimulation (EFS; supramaximal voltage, 0.5 ms; for 10 s) in all three groups evoked only twitch responses at 1-5 Hz, which were inhibited by piridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), a 2PX receptor antagonist. At 10-50 Hz the twitch responses were followed by sustained contractile responses, which were inhibited by prazosin, an α1-adrenoceptor antagonist. These responses were markedly enhanced at higher frequencies in hibernated tissues. At 10°C, EFS evoked only the PPADS-sensitive transient responses in all the three groups, and this was markedly enhanced in hibernated tissues. At 34°C, a single stimulus evoked a PPADS-sensitive excitatory junction potential (EJP) in all three groups but a train of pulses (e.g. ≥0.5) evoked EJPs and prazosin-sensitive sustained depolarizations. These responses were markedly enhanced in hibernated cells. At 10°C, either a single stimulus or a train of stimuli evoked only transient PPADS-sensitive EJPs, which were markedly enhanced in hibernated cells. The contractile responses and electrical membrane responses to exogenous ATP (1-1000 μm) and noradrenaline (0.1-100 μm) were unchanged in the three groups at 34 and at 10°C. These results suggest that during hibernation enhancement of ATP release from the sympathetic perivascular nerves may occur, leading to an efficient means for maintenance of vascular tone and peripheral resistance. Mammalian hibernation is an unusual physiological phenomenon which occurs under stress conditions such as hypothermia, hypoxia or ischaemia. In hibernation, animals undergo a dramatic drop in body temperature and a fall in heart and respiratory rates (Lyman, 1965; Nedergaard & Cannon, 1990; Nurnberger, 1995). Despite this decline, the peripheral vascular resistance increases with deepening hibernation to keep the blood pressure within a reasonable range, and it is thought that the enhancement of the sympathetic mediation is one of the major contributing factors for this regulation (Lyman & O'Briben, 1963; Ralevic et al. 1997; Karoon et al. 1998). Sympathetic perivascular nerves play an important role in the control of peripheral circulation, and it is now widely accepted that in many of these nerves, ATP is a co-transmitter with noradrenaline (NA) (Kennedy et al. 1986; Burnstock, 1995; Thapaliya et al. 1999). Some pathophysiological studies, on the other hand, indicate that the pattern of the relative contribution of adrenergic and purinergic components in the sympathetic perivascular neurotransmission may alter considerably under certain stressed conditions. In canine cutaneous veins exposed to a cold environment, a reflex enhancement of sympathetic tone is blocked by P2-purinoreceptor desensitization, enabling the prevention of excessive heat loss (Flavahan & Vanhoutte, 1986). In the hypertensive rat tail artery and the induced hypertensive rabbit saphenous artery, increased transmitter release (predominantly ATP) has been claimed (Vidal et al. 1986; Bulloch & McGrath, 1992). Thus, our interest is focused on determining whether the relative contribution of the adrenergic and purinergic involvement in sympathetic perivascular neurotransmission is (1) altered during hibernation in the peripheral resistance vessels, and (2) varies with temperature. We have used microelectrode techniques to investigate the fast excitatory junction potentials (EJPs) since in many isolated arteries it is this response which provides the material evidence for the purinergic involvement in the most accurate way (McLaren et al. 1995; Brock & Cunnane, 1999; Thapaliya et al. 1999). Besides, we have considered that it is indispensable to investigate responses under hypothermic condition (10°C) as well as under normothermic conditions to compare hibernating and non-hibernating animals, since the body temperature of the hibernating hamster remains between 8 and 12°C (Lyman, 1965; Ralevic et al. 1997; Karoon et al. 1998). A preliminary account of these data has been presented previously (Saito et al. 2000).

The present trial examined the possibility that diazepam (DZP, 1 mg/kg) induces hyperphagia by acting on the dopaminergic system. Quinpirole (QP), dopamine D-2 receptor agonist, was used for this purpose. Mice fasted for 24 hr were treated with QP 1 (QP-1) or 2 (QP-2) mg/kg 30 min prior to termination of the starvation. DZP was given to untreated mice and half of the QP-1 and QP-2 treated mice 10 min before the termination of the starvation. Food consumed during six 30 min intervals (30 min-feeding), food consumed for 3 hr (total feeding), time required to enter the room containing food by passing through a maze with four multiple routes (time to banquet), latent period to commencement of eating food after entering the banquet room (latent period), and feeding frequency for the 30 min intervals as well as for 3 hr were measured. DZP stimulated feeding, shortened the latent period without affecting the time to banquet and increased the feeding frequency. The hyperphagic effect was restricted to the first 30 min interval only. Both QP-1 and QP-2 first reduced, then progressively stimulated, and finally reduced feeding without modifying total feeding, thus making a bell-shaped profile. They also prolonged both the time to banquet and the latent period, and reduced the feeding frequency of the first 30 min interval but not that for 3 hr. Both QP-1 and QP-2 canceled all the effects of DZP. These results imply that dopamine D2 receptor is involved in the induction of hyperphagia by DZP.

We investigated the effects of motor activities on transmitter release in mouse nerve-muscle preparations of the diaphragm muscle (DPH), extensor digitorum longus muscle (EDL), and soleus muscle (SOL). Mice were divided into a control group, a motor-restricted (RST) group, and a motor-compelled (CMP) group. The quantal content (m) of endplate potentials was measured intracellularly. In DPH the motor activity was unaffected. In the CMP group the m value of the EDL group increased with increases in the cooperativity of Ca2+ in transmitter release. Compared with the CMP group, the SOL of the RST group had a smaller m value with increases in the cooperativity of Ca2+ in transmitter release. These results suggest that motor activities can influence neuromuscular activity specific to different systems, however, the motor compulsion specifically activated the function of EDL and the motor restriction activated the function of SOL, and these effects might lead to altered activity of the release of transmitter quanta in motor nerve terminals of mice.

Chronic exposure to cold increases the growth of brown adipose tissue and the resistance to more severe cold, thus improving thermogenesis. The present study examined the possibility that dietary compounds can modify cold acclimation. Adenosine (ADO) or adenine (ADE) were administered in drinking water (0.05%, w/v) for 5 weeks to male ddY strain mice from 4 weeks of age. At 5 weeks of age, the mice were exposed to 4°C for 4 weeks. After termination of this period, the interscapular brown adipose tissue (IBAT) and rectal temperature on acute exposure to severe cold (–20°C) for 60 min were measured. Chronic exposure to cold increased the weight of IBAT and made mice resistant to a fall in rectal temperature on exposure to severe cold. The growth of IBAT and improvement in thermogenesis can be used as a cold acclimation profile in ddY mice. The growth of IBAT was selectively prevented by ingestion of ADO. The improvement in thermogenesis was reduced, but only a little, by the ingestion of either ADO or ADE. Thus, growth of brown adipocytes might not be necessary for cold acclimation in mice.

Fecal survey of Eimeria oocysts and parasite eggs was conducted for 219 fecal samples of free-ranging Grus japonensis in Kushiro district in Hokkaido in April 2003. Positive rate and mean oocysts (or eggs) per gram in positive samples were 26% (57/219) and 8.8 (0.2-136) in oocysts of Eimeria reichenowi, 18.3% (40/219) and 320 (100-1,000) in trematode eggs, 0.1% (2/219) and 0.2 (0.2-0.3) in eggs of Nematoda A, and 4.1% (9/219) and 0.8 (0.2-3.6) in eggs of Nematoda B, respectively.

Silicon precipitation inside a glass is an important technique for silicon photonics. We successfully precipitated silicon inside silicate glasses containing an Al metal film using femtosecond laser irradiation. First, the Al-inserted sandwiched glass was fabricated by the direct bonding method. The results of a tensile test indicated that the adhesive strength of the sandwich structure reached approximately 4 MPa. Next, femtosecond laser pulses were focused at the Al/glass interface in the sandwich structure. A transmission electron microscopy photograph at the focus of the laser showed that the Al particles were dispersed into the glass substrate to a depth of approximately 2 microm from the initial Al layer. In addition, Raman spectra indicated that silicon had formed at the interface between the glass and Al film after the laser irradiation. The morphology or the particle size of the precipitated silicon was successfully modified by changing the repetition rate or the pulse energy of the laser.

We have succeeded in silicon (Si) precipitation inside a glass/aluminum (Al) sandwich structure via photoinduced reaction using femtosecond (fs) laser irradiation. The sandwich structure was fabricated by direct bonding below 573 K. Raman spectra at the photomodified area indicated that Si crystals formed at the interface between the glass and metallic Al after the laser irradiation. In addition, the particle size of the precipitated Si could be changed by changing the pulse energy of the laser. Furthermore, we have also focused the laser pulses on Fe-Si film to trigger crystallization and phase transformation of FexSiy at the interface between Fe/Si multilayer and glass.

1. The effects of feeding conditions on the sensitivity to the effect of tubocurarine (dTc) in vitro were compared among various nerve-muscle preparations from mice. The mice were fed under conditions that restricted or compelled their movement for 64 days and controls were fed conventionally. 2. The sensitivity to the effect of dTc differed considerably among preparations. It was much higher in the sciatic nerve-extensor digitorum longus muscle (EDL), moderately higher in the sciatic nerve-soleus muscle (SOL) and lower in the phrenic nerve-diaphragm (DPH) in control mice. 3. The order of the sensitivity was not altered by either type of conditioning. Constant restriction of movement or compelled movement did not modify the sensitivity of DPH to the effect of dTc in vitro. 4. Compulsion facilitated the sensitivity in both SOL and EDL. Restriction selectively increased the sensitivity of EDL. Both types of conditioning selectively and significantly reduced twitch development in EDL. 5. These results indicate that the sensitivity to dTc of neuromuscular transmission reflects constant states of motor activity.

The effects of phenylalanine (PHE) and tyrosine (TYR) on cold acclimation were studied in mice of the ddY strain. At 5 weeks of age, mice were maintained at 4 degrees C for 4 weeks. Test groups of mice were supplied with a 0.05% (w/v) solution of PHE or TYR as drinking water in addition to water. We measured changes in body weight, intake of water and food, rectal temperature upon acute exposure to -20 degrees C, weight of interscapular brown adipose tissue (BAT) and the levels of glucose, nonesterified fatty acids (NEFA) and 3H-butyrate in the blood prior to and after exposure to the lower temperature of -20 degrees C. Chronic exposure to cold (4 degrees C) reduced body-weight gain for the first two weeks but weight gain recovered within the next two weeks. PHE and TYR partially inhibited the gain in body weight under exposing to cold. TYR reduced the gain of body weight under room temperature. Exposure to cold stimulated the daily consumption of food. Both PHE and TYR somewhat enhanced the food intake when exposed to cold. Exposure to cold rendered mice resistant to severe cold (-20 degrees C). Both PHE and TYR did not reduce this resistance except in the early state in the case of TYR. Exposure to cold increased the weight of BAT but both PHE and TYR prevented this increase. The effect of TYR was determined in the case fed under room temperature. Cold exposure changed the utilization of glucose, NEFA and 3H-butyrate in the blood when exposed to severe cold (-20 degrees C). Both PHE and TYR prevented the change in NEFA. It remains to be confirmed whether the growth of brown adipocytes is always necessary when mice acclimate to cold.

The effects of adenosine (ADO) and adenine (ADE) on cold acclimation were studied in mice of the ddY strain. At 5 weeks of age, mice were maintained at 4 degrees C for 4 weeks. Test groups of mice were supplied with a 0.05% (w/v) solution of ADO or ADE as drinking water in addition to water. We measured changes in body weight, intake of water and food, rectal temperature upon acute exposure to -20 degrees C, weight of interscapular brown adipose tissue (BAT) and the levels of glucose, nonesterified fatty acids (NEFA) and 3H-butyrate in the blood prior to and after exposure to the lower temperature of -20 degrees C. Chronic exposure to cold (4 degrees C) reduced body-weight gain for the first two weeks but weight gain recovered within the next two weeks. ADO partially but selectively inhibited the gain in body weight. This effect was marked in mice maintained at room temperature. Such an inhibitory effect was the case of ADE in the room temperature. Exposure to cold stimulated the daily consumption of food. ADO further and selectively enhanced food intake, but ADE enhanced that in room temperature. Cold increased in daily intake of water. Both ADO and ADE accelerated the intake of water. This effect was marked in mice maintained at 4 degrees C and the effect of ADE was considerable. Mice chose to drink the water that contained ADO, while the choice of water that contained ADE was apparent only during exposure to cold. Exposure to cold rendered mice resistant to severe cold (-20 degrees C). ADO and ADE partially reduced this resistance. Exposure to cold increased the weight of BAT but ADO selectively prevented this increase. Cold did not change the levels of the various compounds measured in the blood. In mice exposed to 4 degrees C for 4 weeks, acute exposure to severe cold decreased the glucose level and increased the levels of NEFA and 3H-butyrate. ADO selectively prevented the changes in the levels of NEFA and 3H-butyrate. It remains to be confirmed whether the growth of brown adipocytes is always necessary when mice acclimate to cold.

1. The effects of several conditions and agents on the twin-pulse facilitation of the release of transmitter at the mouse neuromuscular junction in low-Ca2+ high-Mg2+ bathing solutions were examined. 2. Twin-pulses gave two endplate potentials (epps) with first (m1) and second (m2) quantal contents. The ratio of m2/m1 was taken as a measure of the degree of facilitation. 3. The mean value of this ratio was >1. Individual ratios fluctuated widely at junctions with smaller values of m1 but were focused around 1 at junctions with larger values of m1. Thus, some populations of junctions with smaller values of m1 contributed to an increment in the mean ratio. 4. The mean ratio was virtually constant irrespective of changes in the spontaneous and evoked release of transmitter at temperatures between 20 and 36°C and at external concentrations of Ca2+ from 0.4 to 0.8 mM. 5. 4-Aminopyridine(4-AP) slightly but significantly increased this ratio with increases in m1 and m2 at temperatures of 24 and 36°C. Ouabain slightly but significantly reduced the ratio, with increases in m1 and m2. The steadiness of the ratio was maintained in the presence of caffeine, high K+, neomycin or omega-conotoxin irrespective of changes in m1 and m2, except in the case of omega-contoxin. 6. Spontaneous output at 36°C increased in the presence of 4-aminopyridine, ouabain, caffeine, high K+ or neomycin. 7. These results indicate that maintenance of a stable value of the ratio of m2 to m1 is a dominant feature. The enhancement of the facilitation by 4-AP implies a contribution of another depolarization-dependent factor which may include a prolongation of the period of inflow of Ca2+ at the terminal.

1. The effects of a reduction in temperature were examined on evoked and spontaneous release of transmitter quanta and on presynaptic negative signals, blocked by Cd2+, measured externally at neuromuscular junctions in mouse diaphragm muscles in low-Ca2+, high-Mg2+ Krebs-Ringer solutions. 2. The evoked release was enhanced with lowering of the temperature, whereas the extent of spontaneous release was reduced. Cooperativity of Ca2+ in the evoked release was slightly reduced by lowering the temperature. 3. The presynaptic negative signals increased in duration with lowering of the temperature. 4. These results support the hypothesis that the effect of a reduction in temperature reflects the improved efficacy of the calcium-mediated mechanism of transmitter release, manifested as a prolongation of the inflow of Ca2+. The process involved in the evoked release is probably attributable to an almost passive mechanism.

To investigate how compensatory responses develop after the onset of inhibition of NPY signaling, we examined the effect of continuous intracerebroventricular (ICV) injection of neutralizing NPY antibodies (NPY-ab) on daily and fast-induced food intake in mice. A single ICV injection of NPY-ab reduced food intake in fasted mice. In contrast to a single injection, continuous ICV injection of NPY-ab for 13 days increased fast-induced food intake, although daily food intake was unaffected by continuous administration of NPY-ab. Immunohistochemistry indicated that the expression of NPY protein increases in the arcuate nucleus, lateral hypothalamic area, and paraventricular nucleus 7 days after onset of continuous NPY-ab infusion and remains at an elevated level, whereas the expression of the NPY Y1 receptor transiently increases in the same areas for 3 days and then gradually decreases. Similar results were obtained for the expression of NPY and NPY Y1 receptor mRNA. The mRNA level of agouti-related protein, another orexigenic neuropeptide, also increased in parallel with NPY, whereas that of pro-opiomelanocortin did not change over the 13 days of the NPY-ab administration. These results suggest that chronic central inhibition of NPY immediately activates orexigenic signaling in first-order hypothalamic neurons and that this compensatory mechanism normalizes the regulation of feeding and energy expenditure to maintain energy homeostasis. On the other hand, in mice that have acquired this compensation, fast-induced food intake further increases even after the energy deficit is corrected because of the dominant orexigenic signal.